体外对牛冠状病毒复制具有抑制效应藏药的筛选

doi:10.11843/j.issn.0366-6964.2025.02.031

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (2): 826-838.doi: 10.11843/j.issn.0366-6964.2025.02.031

体外对牛冠状病毒复制具有抑制效应藏药的筛选

赵龙¹(), 林静怡¹, 豆薇¹, 徐婷萱¹, 顾庆云², 高海慧¹^,³, 李生庆⁴^,*(), 郭抗抗¹^,*()

1. 西北农林科技大学动物医学院, 杨凌 712100
2. 西藏职业技术学院西藏兽药重点实验室, 拉萨 850030
3. 宁夏农林科学院动物科学研究所, 银川 750002
4. 青海省畜牧兽医科学院, 西宁 810016

收稿日期:2024-04-02 出版日期:2025-02-23 发布日期:2025-02-26
通讯作者: 李生庆,郭抗抗 E-mail:448069517@qq.com;lsq.8008@163.com;guokk2007@nwsuaf.edu.cn
作者简介:赵龙(1994-)，男，四川德阳人，博士生，主要从事动物疫病致病机制和防控技术研究，E-mail: 448069517@qq.com
基金资助:
西藏自治区科技计划项目(XZ202101YD0001C)；宁夏回族自治区重点研发计划(2021BEF03005)；青海省科技计划项目(2022-NK-118)

In vitro Screening of Tibetan Medicine with Inhibitory Effects on Bovine Coronavirus Replication

ZHAO Long¹(), LIN Jingyi¹, DOU Wei¹, XU Tingxuan¹, GU Qingyun², GAO Haihui¹^,³, LI Shengqing⁴^,*(), GUO Kangkang¹^,*()

1. College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
2. Tibet Veterinary Medicine Key Laboratory of Xizang Vocational and Technical College, Lhasa, Tibet 850030, China
3. Institute of Animal Science, Ningxia Academy of Agricultural and Forestry Sciences, Yinchuan, Ningxia 750002, China
4. Qinghai Academy of Animal and Veterinary Sciences, Xining, Qinghai 810016, China

Received:2024-04-02 Online:2025-02-23 Published:2025-02-26
Contact: LI Shengqing, GUO Kangkang E-mail:448069517@qq.com;lsq.8008@163.com;guokk2007@nwsuaf.edu.cn

摘要/Abstract

摘要：

本研究旨在筛选出对牛冠状病毒(BCoV)体外复制具有抑制效应的藏药。构建BCoV核衣壳(N)蛋白原核表达载体，诱导表达后将纯化的蛋白分别免疫小鼠和兔，获得鼠抗和兔抗N蛋白多克隆抗体，建立用于测定BCoV复制的Western blot和间接免疫荧光法(IFA)；水煎法获得21种候选藏药的水提物，CCK-8法测定水提物对人结直肠腺癌细胞(HCT-8)的最大安全浓度，以BCoV感染HCT-8细胞为研究对象，分别设置BCoV感染组、藏药水提物处理组、利巴韦林处理组，在BCoV感染24 h后分别应用实时荧光定量PCR、Western blot、IFA测定细胞中BCoV复制量，统计分析藏药水提物对病毒复制的抑制作用，筛选对BCoV具有显著抑制作用的藏药。结果显示：分别制备鼠抗与兔抗BCoV N蛋白多克隆抗体，建立了检测BCoV的Western blot与IFA方法；21种藏药中的15种具有显著抑制BCoV复制效应(P＜0.01)，其中青藏大戟、诃子、瑞香狼毒3种藏药对BCoV复制的抑制作用优于利巴韦林。本研究筛选到15种对BCoV体外复制具有抑制效应的藏药，其中青藏大戟、诃子、瑞香狼毒3种藏药抑制作用效果显著，为BCoV防治药物的开发奠定了基础。

关键词: 牛冠状病毒, 核衣壳蛋白, 多克隆抗体, 藏药, 病毒复制

Abstract:

This study was aimed at screening Tibetan medicines exerting inhibitory effects on the in vitro replication of Bovine coronavirus (BCoV). To achieve this, a prokaryotic expression vector encoding the BCoV nucleocapsid (N) protein was constructed. Following induced expression, the purified N protein was used to immunize mice and rabbits, resulting in the production of mouse anti-N protein polyclonal antibodies and rabbit anti-N protein antibodies. Western blot and indirect immunofluorescence assay (IFA) techniques were subsequently developed to assess BCoV replication. The water extract of 21 candidate Tibetan medicines was obtained by water decoction method. The maximum safe concentration of water extract on human colorectal adenocarcinoma cells (HCT-8) was determined by CCK-8. HCT-8 cells infected with BCoV were used as the research object, and BCoV infection group, Tibetan medicine water extract treatment group and ribavirin treatment group were set up respectively. After 24 h of BCoV infection, real-time fluorescence quantitative PCR, Western blot and IFA were used to determine the replication of BCoV in cells. The inhibitory effect of Tibetan medicine on virus replication was statistically analyzed, and Tibetan medicine with significant inhibitory effect on BCoV was screened. The polyclonal antibodies of mouse anti-BCoV N protein and rabbit anti-BCoV N protein were prepared respectively, and the Western blot and IFA methods for detecting BCoV were established. Among the 21 Tibetan medicines, 15 had significant inhibitory effects on BCoV replication (P < 0.01). Among them, the inhibitory effects of three Tibetan medicines, Euphorbia altotibetica Paulsen, Terminalia chebula Retz., and Stellera chamaejasme L. on BCoV replication were better than those of ribavirin. In this study, 15 Tibetan medicines with inhibitory effects on BCoV replication in vitro were screened. Among them, three Tibetan medicines, Euphorbia altotibetica Paulsen, Terminalia chebula Retz., and Stellera chamaejasme L. had significant inhibitory effects, which laid a foundation for the development of BCoV prevention and treatment drugs.

Key words: bovine coronavirus, N protein, polyclonal antibody, Tibetan medicine, viral replication

中图分类号:

赵龙, 林静怡, 豆薇, 徐婷萱, 顾庆云, 高海慧, 李生庆, 郭抗抗. 体外对牛冠状病毒复制具有抑制效应藏药的筛选[J]. 畜牧兽医学报, 2025, 56(2): 826-838.

ZHAO Long, LIN Jingyi, DOU Wei, XU Tingxuan, GU Qingyun, GAO Haihui, LI Shengqing, GUO Kangkang. In vitro Screening of Tibetan Medicine with Inhibitory Effects on Bovine Coronavirus Replication[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 826-838.

图/表 7

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药物名称 Name of medicine	干粉质量/g Weight of dry powder	药物初始浓度/(mg·mL^-1) Initial drug concentration	最大安全浓度/(mg·mL^-1) Maximum safe concentration
独一味	13.22	55.80	3.49
蓝花荆芥	9.32	38.75	4.84
绿萝花	11.35	91.91	11.49
藏木香	14.66	41.12	20.56
盘花垂头菊	17.11	45.96	1.44
诃子	14.90	1.30	0.16
青藏大戟	18.38	3.95	0.12
乌奴龙胆	12.72	223.21	111.61
短穗兔耳草	15.35	1.30	0.33
五脉绿绒蒿	9.88	208.64	6.52
长松萝	17.26	11.88	1.48
余甘子	15.77	4.88	0.31
蒺藜	10.62	71.25	17.81
铁棒锤	18.39	15.63	0.98
螃蟹甲	16.715	7.42	1.86
甘青青兰	13.99	39.06	4.88
川西獐牙菜	13.24	39.06	9.77
甘青乌头	16.71	14.47	3.67
矮紫堇	17.23	82.25	2.57
瑞香狼毒	15.12	56.88	7.17
虎耳草	12.73	18.59	1.16
利巴韦林	0.10	0.38	0.024

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体外对牛冠状病毒复制具有抑制效应藏药的筛选

In vitro Screening of Tibetan Medicine with Inhibitory Effects on Bovine Coronavirus Replication

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